Much of the difficulty in treating solid tumors lies in the fact that tumor cells are often hypoxic, making delivery of chemotherapeutic drugs and radiation difficult, and hypoxia in itself can directly contribute to tumor metastasis. Cancer cells also cycle in and out of hypoxia on several timescales and with complex dynamics, which has compounded the difficulty in studying these processes. To this end, I propose to engineer a set of synthetic biological devices with the capability of detecting and remembering hypoxic events in the life of a cancer cell. Specifically, I will generate a set of hypoxia- inducible transcription factors with the ability to bind to and stimulate expression of a second reporter molecule. The reporter molecule will be part of a positive feedback loop, upregulating its own expression and providing a continuously present reporter with an output that can be read at any point during the lifetime of the cell, effectively 'remembering'the past hypoxic experience. These devices will then be coupled to a cell cycle based counting device. The proposed counting device will be associated with cell cycle specific proteins to allow the number of cell divisions to be monitored, and by linking the counting device to the hypoxia inducible transcription factors, the number of cell divisions following a hypoxic event can be determined. The results of these experiments will have relevance for understanding how hypoxia affects the behavior of cancer cells, as well as how radiation and chemotherapeutic treatments are altered. Furthermore, these experiments will provide a well-characterized set of tools which will be readily available for use by the research community to investigate a wide variety of human disease-related questions. PUBLIC HEALTH RELEVANCE: The proposed research project involves the engineering of a set of synthetic biological devices with the ability to register transient hypoxic events in the life of cancer cells. The dynamic nature of the hypoxic environment within tumor cells has proven to be a major obstacle in the understanding and treatment of solid tumors. Providing a method to monitor the history of hypoxic events a cell experiences which can be related to the cell's response to radiation and chemotherapy, vascularity, and proliferative capacity will allow these complex interactions to be more fully understood. Additionally, the modularity of the devices proposed here will also allow their facile modification for application to a wide variety of biological questions.